Open AccessFunctional role of cysteine-146 in Escherichia coli thymidylate synthase.
Author(s) -
I K Dev,
Barbara B. Yates,
Jenny Leong,
Walter S. Dallas
Publication year - 1988
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.85.5.1472
Subject(s) - serine , alanine , cysteine , chemistry , stereochemistry , threonine , dissociation constant , biochemistry , enzyme , escherichia coli , amino acid , mutant , thymidylate synthase , binding site , biology , fluorouracil , receptor , genetics , chemotherapy , gene
Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.